Alterations of CpG island DNA hypermethylation and chromatin modification have been widely documented in human cancers (1,2). DNA methylation changes are not only detectable in tumors, but also in blood, as tumor-derived DNA is released into the bloodstream due to tumor necrosis and apoptosis (3,4). Cancer-specific DNA methylation alterations present in cancer tissues and blood of cancer patients can serve as diagnostic markers for risk assessment, progression, early detection, treatment prediction and monitoring (5).
The sensitive detection of specific DNA methylation patterns occurring at very low abundance presents technological challenges that are distinct from the challenges of determining the sequence of consecutive methylation states at single base-pair resolution in individual DNA molecules. The former requires high signal-to-noise ratio, and generally relies on methylation-specific PCR priming (MSP) (6), with optional further enhancement by methylation-specific probing (MethyLight) (7), whereas high-resolution sequencing requires low-sensitivity methylation-independent priming, combined with separation of PCR products for sequence analysis. This separation has traditionally been accomplished by a plasmid cloning step in E. coli prior to sequencing (8).
MethyLight is a quantitative, TaqMan-based real-time PCR assay for measuring methylation levels at a known CpG-containing DNA sequence using bisulfite-converted DNA as a substrate. A high specificity for methylated DNA is attained because both methylation-specific priming and probe annealing events are required to occur.
There is a pronounced need in the art for improved methods for bisulfite genomic sequencing. There is a pronounced need in the art for sensitive detection of aberrant DNA methylation in, for example, cancer patients. There is a pronounced need in the art for sensitive detection of aberrant DNA methylation to provide for improved cancer diagnosis and/or surveillance. There is a pronounced need in the art for novel methylation assay methods that have sufficient resolution to identify and quantify single methylated DNA molecules in a background of unmethylated or competitive genomic DNA.